Cell block cassette device

ABSTRACT

A tissue cassette assembly includes a housing having a recess formed therein, and a compressible reservoir disposed partially or wholly inside of, or otherwise attached in fluid communication with, the housing recess, the compressible reservoir containing a tissue embedding material. The tissue cassette further includes a port disposed in the housing, the port in fluid communication with the compressible reservoir at one end and terminating in a sample cavity at another end. During operation, the compressible reservoir is compressed or squeezed to release the tissue embedding material into the sample cavity containing the biological sample.

FIELD OF THE INVENTION

The present invention relates to devices for embedding biologicalsamples in a solid or semi-solid medium.

BACKGROUND

A technique, commonly referred to as a cell block preparation, has beenused for preparing embedded biological samples for further analysis. Thecell block procedure immobilizes cells or small tissue fragments in asolid or semi-solid support, typically paraffin or wax. Thin sections ofthe cell block are then cut with a microtome and the sections mountedonto a microscope slide for examination. The resulting sections from thecell block display diagnostic information in a manner that complementsdirect deposition techniques.

The cell block preparation method requires that the cell fragments be“embedded” in a solid or semi-solid medium, most commonly paraffin wax.“Embedding” typically requires the following steps: (1) all watermolecules must be removed from the cells, typically by alcohol (water ismiscible with alcohol); (2) all alcohol must then be removed, as well asall fatty substances, and replaced typically by xylene (xylene ismiscible with alcohol but not water); (3) the xylene must be removed andreplaced with wax (wax is miscible with xylene but not with mostalcohols or water); and (4) the cells in molten wax must then bemanually organized and hardened on the underside of a tissue cassette sothat a section of the wax block with the embedded tissue can be cutusing a microtome.

U.S. Pat. No. 6,913,921 discloses methods and apparatuses for preparingembedded samples. The '921 patent, which is incorporated by reference asif set forth fully herein, discloses placing a biological sample on afilter, washing it with alcohol and xylene, and embedding the sample inparaffin wax. A drawback of the disclosed method is the use of a bulkparaffin reservoir, which requires special heating and pumping systems.

SUMMARY OF THE INVENTION

Disclosed herein is a tissue cassette assembly comprising a reservoir; asample cavity; and at least one delivery passage in communication withthe reservoir and the sample cavity. Also disclosed herein is areservoir adapted to fit wholly or partially within, or otherwiseconnect to, a tissue cassette assembly, the reservoir being in fluidcommunication with the sample cavity. Furthermore, disclosed herein is amethod of embedding biological material in tissue embedding material.The method includes the steps of: obtaining a tissue cassette assemblycomprising a sample cavity and a reservoir, where the reservoir containstissue embedding material, and where the reservoir is in fluidcommunication with the sample cavity through a passageway or port;placing the biological material within the sample cavity; squeezing thereservoir, thereby releasing the tissue embedding material from thereservoir into the sample cavity through the passageway, therebyembedding the biological material within the tissue embedding material.

In one embodiment, the tissue cassette assembly includes a housing orbody that includes a recess for receiving and holding a compressiblereservoir. The compressible reservoir is configured to contain a tissueembedding material such as, for instance, paraffin. The compressiblereservoir is fluidly coupled to a sample cavity that is disposed insidethe housing. The compressible reservoir may be fluidly coupled to thesample cavity using one or more channels or tubes. In addition, thetissue cassette assembly may include an optional valve interposedbetween the compressible reservoir and the sample cavity. The valve mayopen or permit the passage of tissue embedding material after asufficient threshold amount of compressive force is applied to thecompressible reservoir. The tissue cassette assembly may have multiplereservoirs (e.g., upper and lower) located in separate recesses of thehousing. The compressible reservoir may be permanently attached orotherwise secured to the tissue cassette assembly. Alternatively, thecompressible reservoir may be attached or inserted into (whethermanually or automatically) the housing body (e.g., for example, insertedafter manufacture).

In another embodiment, a tissue cassette assembly includes a housinghaving at least one recess therein, and a compressible reservoirdisposed inside the at least one recess of the housing, the compressiblereservoir configured to contain tissue embedding material. The tissuecassette further includes at least one port disposed in the housing, theat least one port being in fluid communication with the compressiblereservoir at one end and terminating in a sample cavity at another end.

In still another embodiment, a method of embedding biological materialin tissue embedding material includes providing a tissue cassetteassembly of the type described above. Biological material is then placedinside the sample cavity. The reservoir is then compressed (e.g.,squeezed) to release the tissue embedding material from the compressiblereservoir into the sample cavity so as to embed the biological materialwithin the tissue embedding material. In one aspect, the biologicalmaterial is placed on a piece of filter paper or similar porous mediathat is fixedly secured inside the sample cavity. For example, thefilter paper may rest overtop a metallic plate having a plurality ofapertures or holes therein.

In still another embodiment, a reservoir for containing a tissueembedding material includes a compressible container having at least oneopening, the compressible container configured to contain a volume oftissue embedding material therein, the compressible container beingfurther configured to fluidly couple with at least one port in a tissuecassette assembly, the at least one port being in fluid communicationwith a sample cavity disposed within the tissue cassette assembly.

In yet another embodiment of the invention, a tissue cassette assemblyincludes a housing having at least one recess therein, the at least onerecess containing a tissue embedding material. A moveable piston isdisposed inside the at least one recess of the housing, the moveablepiston configured to apply pressure to the tissue embedding material inthe recess. The tissue cassette further includes at least one portdisposed in the housing, the at least one port being in fluidcommunication with the tissue embedding material in the recess at oneend and terminating in a sample cavity at another end.

BRIEF DESCRIPTION OF THE DRAWINGS

It will be appreciated that the drawings are not necessarily to scale,with emphasis instead being placed on illustrating the various aspectsand features of embodiments of the invention, in which:

FIG. 1A depicts a top view of an embodiment of a cell block devicehaving one passageway or port coupling the reservoir and the samplecavity.

FIG. 1B depicts a top view of another embodiment of a cell block devicewherein there is a single passageway or port located at the side of thereservoir between the reservoir and the sample cavity (e.g., cell blockchamber).

FIG. 1C depicts a top view of another embodiment of a cell block devicewherein there are two passageways or ports connecting the reservoir andthe sample cavity.

FIG. 2 depicts a bottom view of a cell block device having multiple(upper and lower) reservoirs. Only one such reservoir can be seen inFIG. 2.

FIG. 3A depicts a cross-sectional view of an embodiment of a cell blockdevice according to one embodiment, wherein a reservoir is located atthe top side of the cell block.

FIG. 3B depicts a cross-sectional view of an embodiment of a cell blockdevice, wherein a first reservoir is located at the top side of the cellblock and a second reservoir is located at the bottom side of the cellblock.

FIG. 4 depicts a cross-sectional view of an embodiment of a cell block,wherein the reservoir includes a moveable piston.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Embodiments of the present invention are described below. It is,however, expressly noted that the present invention is not limited tothese embodiments, which are provided by way of example and notlimitation. Aspects of the present invention are directed to a cellblock cassette assembly having a reservoir or a pouch that can contain aliquid or semi-liquid substance, such as alcohol, xylene, or a tissueembedding material, such as liquefied wax, paraffin, paraffin withadditives, plastics, and the like. In some embodiments, the tissueembedding material is paraffin. Once the pouch or reservoir is squeezed,either manually or by an instrument, the contents of the pouch orreservoir is emptied into a sample cavity, which contain a biologicalsample, such as cells or tissue. The use of the pouch or reservoirembedded within the cell block cassette removes the need for an externalreagent flask. Some of the benefits of the present invention include theconvenience of not needing to load bulk wax and virtually eliminatingthe problems of contamination of the wax during handling and thepossibility of using waxes that are not qualified to work in theapparatus. In addition, the cell block cassette having the reservoir mayoptionally be disposable. In these embodiments, maintenance of the waxpumping system and possible problems with buildup and clogs is virtuallyeliminated.

FIG. 1A shows an embodiment of the tissue cassette 102. The tissuecassette 102 generally includes a housing or body portion 103 thatincludes the various working features of the device. The housing or bodyportion 103 may be formed, at least in part, from a polymer or plasticmaterial. The housing or body portion 103 may be formed as a singleunit. Alternatively, the housing or body portion 103 may be formed frommultiple sub-units that are then subsequently bonded or affixed to forma unitary construction such as that illustrated in FIG. 1A. The tissuecassette 102 includes a sample cavity 104, which is adapted to receivecells, tissues, or other biological samples to be embedded in the solidsupport medium. The sample cavity 104 may be milled or drilled out ofthe housing or body portion 103. As seen in FIGS. 3A, 3B, and 3C, thesample cavity 104 may have support member 105 disposed about theperiphery of the sample cavity 104. For example, the support member 105may be formed as a ring or series or projections that extend radiallyinward from the sample cavity 104. The support member 105 (or pluralityof members 105) may be used to rest a porous member such as a plate oreven filter paper during the embedding process. For example, a metalplate (not shown) having a plurality of holes may be inserted into thesample cavity 104. Filter paper or other porous media may then bepositioned over the metal plate to create the thin layer of cells orbiological tissue needed for the embedding process.

Still referring to FIG. 1A, the sample cavity 104 is cylindrical inshape. However, in other embodiments, the sample cavity 104 can haveother shapes including, for example, cross-sectional shapes of a square,triangle, polygon (e.g., pentagon, hexagon, heptagon, octagon, etc.),oval, or the like. The tissue cassette 102 also includes a pouch orreservoir 106 that is disposed inside a recess 101 that is located inthe housing 103. In alternate embodiments, the reservoir 106 is attachedto the tissue cassette 102, but is not within a recess located in thehousing 103. The reservoir 106 is configured as a compressible orflexible container that holds a tissue embedding material, such as wax,paraffin, paraffin with additives, plastics, and the like. The reservoir106 is constructed from material that is resistant to degradation due toexposure to certain chemicals, such as paraffin, plastics, other waxes,and solvents, such as water, alcohols, and xylene. The constructionmaterial for the reservoir 106 is also resistant to degradation due toexposure to the temperature required to melt the tissue embeddingmaterial and pressure required to remove the tissue embedding materialfrom the reservoir 106. The reservoir 106 may be constructed fromflexible plastic or other polymer material, metal foil, polyester,fluorocarbon polymer, vinyl, and rubber. The reservoir 106 hassufficient strength such that any fluid contained therein will not leakor burst out in response to an applied pressure (except for that portionin communication with the sample cavity 104 described in detail below).

In certain embodiments, the reservoir 106 may be permanently affixed tothe tissue cassette 102. In other embodiments, the reservoir 106 can beremoved and replaced by another reservoir 106. In certain embodiments,the reservoir 106 in filled at a central location, such as a factory ora manufacturing facility, affixed to the tissue cassette 102, andshipped as such. In other embodiments, the reservoir 106 is empty whenshipped and is filled by the end user. In further embodiments, thereservoir 106 is refillable by the end user.

As shown in FIG. 1A, the reservoir 106 is in fluid communication withthe sample cavity 104 through a passageway or port 108. As seen in FIG.1A, the passageway 108 is located at the central region of the reservoir106. In another embodiment, shown in FIG. 1B, the passage 110 is locatedat one side of the reservoir 106. This embodiment is particularly usefulif the contents of the reservoir 106 are emptied through the action of aroller, which rolls from one side to another in a direction generallyperpendicular to the long axis of the tissue cassette 102. In yetanother embodiment, shown in FIG. 1C, two passages, 110 and 112, arelocated at opposite sides of the reservoir 106 and connect the reservoir106 to the sample cavity 104. This embodiment is particularly useful fora more even distribution of the tissue embedding material within thesample cavity 104 and for a more rapid dispensing of the tissueembedding material. FIG. 3A shows a cross-section of the embodimentshown in FIG. 1A.

In some embodiments, as shown in FIGS. 2 and 3, in addition to having areservoir 106 at its top side, the tissue cassette 102 has anotherreservoir 202 at its bottom side. The top and bottom reservoirs 106, 202may be located in separate recesses 101, 101′ formed in the tissuecassette 102. The bottom or lower reservoir 202 is in fluidcommunication with the sample cavity 104 through at least one passageway206. FIG. 2 shows one embodiment in which two passages, 204 and 206,connect the reservoir 202 with the sample cavity 104. FIG. 3B shows across-sectional view of the embodiment shown in FIG. 2.

In some embodiments, the passageways 108, 110, 112, 204, and 206, eachis independently a channel molded in the plastic body of tissue cassette102. For example, the tissue cassette 102 may be formed from twohalves—each half containing a partial channel that, when combined, formthe fluid passageways. In other embodiments, the passages 108, 110, 112,204, and 206, each is independently a tube that connects the reservoir106 or 202 with the sample cavity 104. When the passages are a tube, thetube is constructed from material that is resistant to degradation dueto exposure to certain chemicals, such as paraffin, other waxes, andsolvents, such as water, alcohols, and xylene.

FIG. 4 shows another embodiment of the reservoir 402 in the tissuecassette 102. The reservoir 402 comprises a piston assembly 403 that islocated in a recess 101 of the tissue cassette 102. The piston assembly403 comprises a moveable piston 404 (moveable in the direction of arrowA in FIG. 4). While FIG. 4 illustrates the piston 404 as occupying thetop edge of the reservoir 402, in other embodiments, the piston 404occupies one of the sides of the reservoir 402, while in still otherembodiments, the piston 404 is at the bottom of the reservoir 402. Thepiston assembly 403 further comprises a shaft 406, which connects thepiston 404 to a boss 408.

In some embodiments, the side of the reservoir containing the boss 408,e.g., the top side 410 in FIG. 4, is flexible, thus allowing the boss408 greater freedom of movement. In other embodiments, the shaft 406 isof such length that when the boss 408 is depressed, the piston 404 movesthrough substantially all of the reservoir 402.

When the boss 408 is depressed, the piston 404 moves down (in directionof arrow A) and pushes the contents of the reservoir 402, e.g., paraffinor other tissue embedding material, into the sample cavity 104 through achannel 412. In some embodiments, an optional valve 414 separates thereservoir 402 from the sample cavity 104. When the boss 408 isdepressed, the pressure of the contents of the reservoir 402 causes thevalve 414 to open and allow for the contents of the reservoir 402 tomove to the sample cavity 104. For example, the valve 414 may open oreven break upon application of compressive or squeezing force that isabove a certain threshold level.

In another aspect, the reservoir 106, 202 is adapted to fit within atissue cassette 102, where the reservoir 106, 202 is adapted to containtissue embedding material. In some embodiments, the tissue embeddingmaterial is paraffin. The reservoir 106, 202 may contain tissueembedding material in an amount sufficient to embed a biological samplewithin the sample cavity 104 with tissue embedding material. In certainembodiments, the reservoir 106, 202 is constructed from materialresistant to degradation due to exposure to a condition selected fromthe group consisting of contact with the tissue embedding material,contact with solvents, high temperature required to melt the tissueembedding material, and pressure required to remove the tissue embeddingmaterial from the reservoir. The material may include, for example,flexible plastic, metal foil, polyester, fluorocarbon polymer, vinyl,and rubber.

A method of embedding biological material in tissue embedding materialmay include the steps of obtaining a tissue cassette assembly comprisinga sample cavity 104 and a reservoir 106, 202, where the reservoir 106,202 contains the tissue embedding material, and where the reservoir 106,202 is in fluid communication with the sample cavity 104 through adelivery passageway 108, 110, 112, 204, 206. The biological material isplaced within the sample cavity 104 and the reservoir 106, 202 iscompressed or depressed so as to release the tissue embedding materialfrom the reservoir 106, 202 into the sample cavity 104 through thepassageway 108, 110, 112, 204, 206, thereby embedding the biologicalmaterial within the tissue embedding material.

In certain embodiments, the reservoir 106, 202 contains tissue embeddingmaterial in an amount sufficient to at least partially fill the samplecavity 104 with the tissue embedding material. In further embodiments,the reservoir 106, 202 is constructed from material resistant todegradation due to exposure to the tissue embedding material, solvents,high temperatures required to melt the tissue embedding material, andpressure required to remove the tissue embedding material from thereservoir. In some embodiments, the reservoir 106, 202 is formed from amaterial such as, for instance, flexible plastic, metal foil, polyester,fluorocarbon polymer, vinyl, and rubber.

The reservoir 106, 202 may be permanently affixed to the tissue cassette102. In an alternative embodiment, the reservoir 106, 202 isreplaceable. In certain embodiments, the reservoir 106, 202 isrefillable. As described herein, the delivery passage way 108, 110, 112,204, 206 is a channel constructed within the tissue cassette 102. Inother embodiments, the delivery passage is a tube embedded within thetissue cassette assembly. In some of these embodiments, the tube isconstructed from material resistant to degradation. For example, thetube may be formed from a material that resists degradation in responseto exposure to the tissue embedding material, exposure to solvents, andexposure to elevated temperatures and pressures.

The biological material used in connection with the tissue cassette 102may include a collection of cells or cellular bodies. In certainembodiments, the biological material may also include a tissue sample.For example, the tissue sample may be taken from a certain organ or bodytissue for subsequent imaging and analysis.

As explained herein, the tissue embedding material is contained within areservoir 106, 202. The tissue embedding material may be stored withinthe reservoir 106, 202 in a solid or semi-solid state. In this regard,the tissue embedding material may need to be heated to partially orfully melt the tissue embedding material into the liquid or semi-liquidstate. Once the tissue embedding material is in a liquid or evensemi-liquid state, the reservoir 106, 202 can be compressed (e.g.,squeezed) to transfer the tissue embedding material to the one or moredelivery passageways 108, 110, 112, 204, 206 and into the sample cavity104.

In some embodiments, the method disclosed herein further comprisesplacing a porous plate on the support member 105 (or plurality ofmembers 105). For example, a metal plate (not shown) having a pluralityof holes may be inserted into the sample cavity 104 and held in placevia the one or more support members 105. A piece of filter paper orother porous media may then be positioned over the metal plate to createthe thin layer of cells or biological tissue needed for the embeddingprocess. Once a layer of biological material has been deposited on thefilter paper (or other porous media), the reservoir 106, 202 may besqueezed to deposit the embedding material on top of the biologicalmaterial.

If a tissue cassette 102 of the type disclosed in FIG. 3B is used, themethod may further include the step of squeezing a second reservoir(e.g., 202), thereby releasing the tissue embedding material from thesecond reservoir 202 into the sample cavity 104 through a passageway206, where the tissue embedding material from the second reservoir 202is released below the filter paper (or other porous media).

According to one aspect of the method of embedding a biological samplewithin an embedding media, the method disclosed herein further comprisestreating the biological material sequentially with an alcohol and xyleneprior to the squeezing step.

The invention may be embodied in other specific forms besides and beyondthose described herein. The foregoing embodiments are therefore to beconsidered in all respects illustrative rather than limiting, and thescope of the invention is defined only by the appended claims and theirequivalents, rather than by the foregoing description.

1. A tissue cassette assembly, comprising: a housing; a compressiblereservoir attached to the housing and containing a tissue embeddingmaterial; and a sample cavity disposed in the housing, the sample cavitybeing fluidly coupled to the compressible reservoir.
 2. The tissuecassette assembly of claim 2, wherein the tissue embedding materialcomprises paraffin.
 3. The tissue cassette assembly of claim 1, whereinthe compressible reservoir is constructed from material selected fromthe group consisting of plastic, metal foil, polyester, fluorocarbonpolymer, vinyl, and rubber.
 4. The tissue cassette assembly of claim 1,wherein the compressible reservoir is permanently affixed to the tissuecassette assembly.
 5. The tissue cassette assembly of claim 1, whereinthe compressible reservoir is removable from the housing.
 6. The tissuecassette assembly of claim 1, wherein the delivery passageway comprisesa channel disposed within the housing.
 7. The tissue cassette assemblyof claim 1, wherein the delivery passageway is a tube embedded withinthe housing.
 8. The tissue cassette assembly of claim 1, wherein thereservoir contains a volume of tissue embedding material capable ofsubstantially filling the sample cavity.
 9. The tissue cassette assemblyof claim 1, further comprising a second recess located in housing, thesecond recess containing a compressible reservoir.
 10. A tissue cassetteassembly, comprising: a housing; a compressible reservoir attached tothe housing and containing tissue embedding material; and a portdisposed in the housing, the port in fluid communication with thecompressible reservoir at one end and terminating in a sample cavity atanother end.
 11. The tissue cassette assembly of claim 10, furthercomprising a valve interposed between the compressible reservoir and theport.
 12. The tissue cassette assembly of claim 10, wherein thecompressible reservoir is constructed from material selected from thegroup consisting of plastic, metal foil, polyester, fluorocarbonpolymer, vinyl, and rubber.
 13. A tissue cassette assembly, comprising:a housing; a compressible reservoir attached to the housing andcontaining a tissue embedding material; a moveable piston connected tothe compressible reservoir, the moveable piston configured to applypressure to the tissue embedding material in the compressible reservoir;and a port disposed in the housing, the port in fluid communication withthe tissue embedding material in the compressible reservoir at one endand terminating in a sample cavity at another end.
 14. A reservoir forcontaining a tissue embedding material, comprising: a compressiblecontainer having an opening and containing a volume of tissue embeddingmaterial therein, the opening configured to fluidly couple with a portdisposed in a tissue cassette assembly, the port in fluid communicationwith a sample cavity disposed within the tissue cassette assembly.
 15. Amethod of embedding biological material in tissue embedding material,comprising: providing a tissue cassette assembly comprising a samplecavity and a compressible reservoir in fluid communication with thesample cavity, wherein the compressible reservoir contains tissueembedding material in an amount sufficient to at least partially fillthe sample cavity with the tissue embedding material; placing thebiological material within the sample cavity; compressing the reservoir,thereby releasing the tissue embedding material from the reservoir intothe sample cavity so as to embed the biological material within thetissue embedding material.
 16. The method of claim 15, wherein thecompressible reservoir is constructed from material selected from thegroup consisting of plastic, metal foil, polyester, fluorocarbonpolymer, vinyl, and rubber.
 17. The method of claim 15, wherein thebiological material comprises a plurality of cells.
 18. The method ofclaim 15, wherein the biological material comprises a tissue.
 19. Themethod of claim 15, further comprising heating the compressiblereservoir to melt the tissue embedding material prior to compressing thereservoir.
 20. The method of claim 15, further comprising placing afilter paper within the sample cavity and placing the biologicalmaterial over the filter paper.
 21. The method of claim 15, wherein thetissue embedding material is released on top of the biological material.22. The method of claim 21, further comprising compressing a secondcompressible reservoir, thereby releasing tissue embedding material fromthe second compressible reservoir into the sample cavity.